DNA polymerases having improved labeled nucleotide incorporation properties

ABSTRACT

The present invention relates to mutant DNA polymerases that exhibit reduced discrimination against labeled nucleotides into polynucleotides. The DNA polymerases of the invention have at least one mutation in the nucleotide label interaction region of the enzyme such the mutation results in reduced discrimination against labeled nucleotides. The nucleotide label interaction regions is located at portions of the O-helix, (ii) the K helix, and (iii) the inter O—P helical loop of Taq DNA polymerase or analogous positions in other DNA polymerases. In addition to providing novel mutant DNA polymerases, the invention also provides polynucleotides encoding the subject mutant DNA polymerases. The polynucleotides provided may comprise expression vectors for the recombinant production of the mutant polymerases. The invention also provide host cells containing the subject polynucleotides. The invention also includes numerous methods of using the subject DNA polymerases, including uses for chain termination sequencing and PCR. Another aspect of the invention is to provide kits for synthesizing fluorescently labeled polynucleotides in accordance with the methods of the invention. Kits of the invention comprise a mutant DNA polymerase of the invention and a fluorescently labeled nucleotide that exhibits reduced discrimination with respect to the mutant DNA polymerase in the kit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/794,262, filed Feb. 27, 2001, which is a divisional of U.S.application Ser. No. 09/041,878, filed Mar. 12, 1998, (now U.S. Pat. No.6,265,193) which claims a priority benefit under 35 U.S.C. §119(e) fromU.S. Application No. 60/039,610, filed Mar. 12, 1997, all of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention is related to DNA polymerases having mutations that alterthe ability of the enzyme to incorporate labeled nucleotides into apolynucleotide molecule.

BACKGROUND

DNA polymerase are enzymes that synthesize the formation of DNAmolecules from deoxynucleotide triphosphates using a template DNA strandand a complementary synthesis primer annealed to a portion of thetemplate. A detailed description of DNA polymerases and theirenzymological characterization can be found in Komber, DNA ReplicationSecond Edition, W. H. Freeman (1989).

DNA polymerases have a variety of uses in molecular biology techniquessuitable for both research and clinical applications. Foremost amongthese techniques are DNA sequencing and nucleic acid amplificationtechniques such as PCR (polymerase chain reaction).

The amino acid sequence of many DNA polymerases have been determined.Sequence comparisons between different DNA polymerase have identifiedmany regions of homology between the different enzymes. X-raydiffraction studies have determined the tertiary structures of Klenowfragment, T7 DNA polymerase, and Taq DNA polymerase. Studies of thetertiary structures of DNA polymerases and amino acid sequencecomparisons have revealed numerous structural similarities betweendiverse DNA polymerases. In general, DNA polymerases have a large cleftthat is thought to accommodate the binding of duplex DNA. This cleft isformed by two sets of helices, the first set is referred to as the“fingers” region and the second set of helices is referred to as the“thumb” region. The bottom of the cleft is formed by anti-parallel βsheets and is referred to as the “palm” region. Reviews of DNApolymerase structure can be found in Joyce and Steitz , Ann. Rev.Biochem. 63:777-822 (1994). Computer readable data files describing thethree-dimensional structure of some DNA polymerases have been publiclydisseminated.

Fluorescently labeled nucleotides have greatly simplified and improvedthe utility of many procedures in molecular biology. The use offluorescently labeled nucleotides for labeling polynucleotides insynthesis procedures, has to a large extent replaced the use ofradioactive labeling. Fluorescently labeled nucleotides have been widelyused in DNA sequencing, see Smith et al Nature 321:674-679 (1986), inPCR, and other forms of polynucleotide fragment analysis.

A major problem with using fluorescently labeled nucleotides is theability of DNA polymerases to discriminate against the incorporation offluorescently labeled nucleotides. For example, the inventors havediscovered that in competition assays between a TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein) labeled 2′3′dideoxynucleotide and the corresponding unlabeled dideoxynucleotide, TaqDNA polymerase incorporates the unlabeled dideoxynucleotide into DNA atleast 85 times more frequently than the corresponding unlabelednucleotide. This discrimination between labeled and unlabelednucleotides has profound effects on procedures using DNA polymerases tolabel DNA. For example, much larger amounts of fluorescently labelednucleotide must be used in sequencing reactions. This large amount offluorescently labeled nucleotide is expensive and can generate excessivebackground fluorescence, thereby reducing the yield of sequenceinformation.

In view of the problems arising from the ability of DNA polymerases todiscriminate against the incorporation of fluorescently labelednucleotides, the inventors have developed several novel DNA polymerasesthat have reduced discrimination against the incorporation of one ormore fluorescently labeled nucleotides into DNA.

SUMMARY

Naturally occurring DNA polymerases preferentially incorporate unlabelednucleotides over corresponding fluorescently labeled nucleotides intopolynucleotides. This ability of DNA polymerases to discriminate againstfluorescently labeled nucleotide has undesirable effects on manymolecular biology procedures that require the enzymatic addition offluorescently labeled nucleotides, e.g., labeled dideoxy terminatorsequencing. The present invention relates to mutant DNA polymerases thatexhibit reduced discrimination against fluorescently labeled nucleotidesinto polynucleotides.

The DNA polymerases of the invention have at least one mutation in thenucleotide label interaction region of the enzyme such that the mutationresults in reduced discrimination against fluorescently labelednucleotides. The nucleotide label interaction region of a DNA polymeraseis formed by portions of the 0-helix, (ii) the K helix, and (iii) theinter O—P helical loop of Taq DNA polymerase or analogous positions inother DNA polymerases. Amino acid residues within the nucleotide labelinteraction region as defined by TET (II)•ddC are E520. A531, L522,R523, E524, A525, H526, P527, I 528, V529, E530, K531, I532, R536, E537,R573, Q582, N583, V586, R587, P589, Q592, R593, R595, D610, T612, Q613,E615, R636, D637, T640, F647, V654, D655, P656, L657, R659, R660, T664,E681, L682, A683, I684, P685, E688, F692, Q754, H784, L817, E820, L828,K831, and E832. The sites at R660, T664, and E681 are of prefered sitesfor introducing mutations. In a preferred embodiment of the inventionfor use with fluorescein-type dyes, a mutation is present at position681 converting an E (glutamic acid) to M (methionine), i.e., E681M. In apreferred embodiment of the invention for use withfluorescein-fluorescein energy transfer dyes a mutation is present atposition 657 converting an L (leucine) to a G (glycine). In addition toproviding mutant Taq DNA polymerases having reduced discriminationagainst labeled nucleotides, the invention includes mutants derived froma wide variety of DNA polymerases, both thermostable and otherwise.

In addition to providing novel mutant DNA polymerases, the inventionalso provides polynucleotides encoding the subject mutant DNApolymerases. The polynucleotides provided may comprise expressionvectors for the recombinant production of the mutant polymerases. Theinvention also includes host cells containing the subject polymerasepolynucleotides.

The invention also includes numerous methods of using the subject DNApolymerases. The subject methods involve synthesizing a fluorescentlylabeled polynucleotide by means of a polynucleotide synthesis reactioncatalyzed by a mutant DNA polymerase that has reduced discriminationagainst incorporating labeled nucleotides into polynucleotides. Thesubject methods of polynucleotide synthesis include the step ofextending a primed polynucleotide template with at least one fluorescentlabeled nucleotide, wherein the extension is catalyzed by a DNApolymerase that has reduced discrimination against labeled nucleotidesinto polynucleotides. The subject methods of synthesizing afluorescently labeled polynucleotide may be used in a variety of methodssuch as Sanger sequencing and the polymerase chain reaction (PCR).

Another aspect of the invention is to provide kits for synthesizingfluorescently labeled polynucleotides in accordance with the methods ofthe invention. Kits of the invention comprise a mutant DNA polymerase ofthe invention and a fluorescently labeled nucleotide that exhibitsreduced discrimination with respect to the mutant DNA polymerase in thekit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a computer model of DNA bound to Taq DNA polymerase. Aminoacid residues that form the nucleotide label interaction site arehighlighted in orange. The rest of the polymerase is indicated in green.The template is indicated in blue. The dye moiety of the labelednucleotide is red. The remaider of the labled nucleotide is white.

FIG. 2 is plot of a next nucleotide effect assay.

FIG. 3 is plot of a next nucleotide effect assay.

FIG. 4 is a representation of the structure of the fluorescently labelednucleotide “TET(II)•ddCTP.”

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Terminology

Positions of amino acid residues within a DNA polymerase are indicatedby either numbers or number/letter combinations. The numbering starts atthe amino terminus residue. The letter is the single letter amino acidcode for the amino acid residue at the indicated position in thenaturally occurring enzyme from which the mutant is derived. Unlessspecifically indicated otherwise, an amino acid residue positiondesignation should be construed as referring to the analogous positionin all DNA polymerases, even though the single letter amino acid codespecifically relates to the amino acid residue at the indicated positionin Taq DNA polymerase.

Individual substitution mutations are indicated by the form of aletter/number/letter combination. The letters are the single letter codefor amino acid residues. The numbers indicate the amino acid residueposition of the mutation site. The numbering system starts at the aminoterminus residue. The numbering of the residues in Taq DNA polymerase isas described in U.S. Pat. No. 5,079,352 (Gelfand). Amino acid sequencehomology between different DNA polymerases permits correspondingpositions to be assigned to amino acid residues for DNA polymerasesother than Taq. Unless indicated otherwise, a given number refers toposition in Taq DNA polymerase. The first letter, i.e., the letter tothe left of the number, represents the amino acid residue at theindicated position in the non-mutant enzyme. The second letterrepresents the amino acid residue at the same position in the mutantenzyme. For example, the term “R660D” indicates that the arginine atposition 660 has been replaced by an aspartic acid residue.

The term “discrimination” as used herein refers to the property of a DNApolymerase to preferentially incorporate unlabeled nucleotides overcorresponding fluorescently labeled nucleotides into DNA, i.e., the DNApolymerase discriminates against the fluorescently labeled nucleotide.Preferential incorporation may be measured in an assay in which afluorescently labeled 2′3′ dideoxynucleotide and a correspondingunlabeled 2′3′ dideoxynucleotide compete for incorporation into the samesite of a polynucleotide. An example of such an assay can be found belowin example 2.

The term “reduced discrimination” as used herein refers to reduction indiscrimination against incorporation of a fluorescently labelednucleotides in a mutant DNA polymerase as compared to the parent enzyme.A reduction in discrimination may be described quantitatively byreference to the selectivity assays in Example 2 or reference to otherassays providing for measurement of the same properties of thepolymerase. A reduction in selectivity number as measured by theselectivity assays is a reduction in discrimination and may be expressedby a ratio of selectivity numbers. For example, a mutant DNA polymerasewith a selectivity number of 8 would have a 10-fold reduction indiscrimination when compared with a parent DNA polymerase having aselectivity number of 80.

The term “parent” or “parent enzyme” is used to distinguish a mutant DNApolymerase from the DNA polymerase that the mutant enzyme was derivedfrom. Thus any naturally occurring DNA polymerase may be referred to asparent enzyme. A first DNA polymerase having mutations with respect to anaturally occurring enzyme is also be referred to as a parent enzymewith respect to a second DNA polymerase having additional mutations.

The term “discrimination reducing mutations” refers to mutations in thenucleotide label interaction region of a DNA polymerase that result inreduced discrimination against the incorporation of fluorescentlylabeled nucleotides. The term is used to distinguish mutations in a DNApolymerase, including mutations in the nucleotide label interactionregion, that do not reduce discrimination against fluorescently labelednucleotides from mutations that do reduce discrimination.

The term “nucleotide” as used herein, unless specifically notedotherwise, is used broadly to refer to both naturally occurringnucleotide and a variety of analogs including 2′,3′ dideoxynucleotides.

The term “fluorescein-type dyes” refers to a class of xanthene dyemolecules which include the following fused three-ring system:

where a wide variety of substitutions are possible at each deoxy ringposition. A particularly preferred subset of fluorescein-type dyesinclude the 4,7,-dichorofluoresceins (Menchen). Examples offluorescein-type dyes used as fluorescent labels in DNA sequencingmethods include 6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein(5-FAM), 6-carboxy-4,7,2′,7′-tetrachlorofluorescein (TET),6-carboxy-4,7,2′,4′,5′,7′-hexachlorofluorescein (HEX), 5-(and6)carboxy-4′,5′-dichloro-2′7′-dimethoxyfluorescein (JOE), and5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE). Many times thedesignation -1 or -2 is placed after an abbreviation of a particulardye, e.g., HEX-1. The “-1” and “-2” (or “I” and “II”) designationsindicate the particular dye isomer being used. The 1 and 2 isomers aredefined by the elution order (the 1 isomer being the first to elute) offree dye in a reverse-phase chromatographic separation system utilizinga C-8 column and an elution gradient of 15% acetonitrile/85% 0.1 Mtriethylanmuonium acetate to 35% acetonitrile/65% 0.1 M triethylammoniumacetate.

The term “alkynylamino type linker” refers to an alkynylamino linkerofthe type as described in U.S. Pat. No. 5,047,519 (Hobbs), U.S. Pat.No. 5,151,507 (Hobbs), and U.S. patent application Ser. No. 08/696,808,filed Aug. 13, 1996. Additional alkynylamino type liners are describedin U.S. patent application Ser. No. 08/833,855, filed Apr. 10, 1997.

The term “TET(II)•ddCTP” refers to the fluorescently labeled nucleotideof the structure indicated in FIG. 4.

The term “fluorescence energy transfer dye” refers to dye moietiesjoined by a linker that permits fluorescence energy transfer between thetwo dye moieties. For use in chain termination sequencing, the linker issufficiently small and of the proper shape and orientation to permit aDNA polymerase to incorporate a nucleotide triphosphate labeled with thedye interest. Examples of energy transfer dyes can be found in EuropeanPatent Application No. EP 0 805 140, U.S. patent application Ser. No.08/642,330 (filed May 3, 1996), and U.S. patent application Ser. No.08/726,462 (filed Oct. 4, 1996).

The term “mutation” as used herein refers to a change in amino acidresidue at a specific location of a protein. The change in amino acidresidue is a change defined with respect to a naturally occurringprotein. A protein having a mutation may be referred to as a “mutant”protein.

EMBODIMENTS OF THE INVENTION

The present invention relates to DNA polymerases containing mutationsthat reduce the ability of the polymerase to discriminate against theincorporation of fluorescently labeled nucleotides into polynucleotides.These mutations are in a region of the DNA polymerase molecule referredto herein as “the nucleotide label interaction region.” The nucleotidelabel interaction region is formed by portions of three regions of theDNA polymerase. These three regions are located in (i) the O-helix, (ii)the K helix, and (iii) the inter O—P helical loop of Taq DNA polymeraseor analogous positions in other DNA polymerases. DNA polymerases havingreduced discrimination against fluorescently labeled nucleotides areparticularly useful for chain termination DNA sequencing using 2′3′dideoxynucleotides, i.e., Sanger type sequencing.

Enzyme kinetic experiments (described in examples 2 and 3) performedwith Taq DNA polymerase and fluorescently labeled dideoxynucleotidessupport a theory that Taq DNA polymerase and other DNA polymerases,undergo a conformational shift upon the binding of nucleotides duringDNA synthesis. This predicted conformational shift suggests a set ofamino acid residues that interact with fluorescent labels joined by alinker to the nucleic acid base of a nucleotide, thereby resulting indiscrimination against nucleotides that are fluorescently labeled. Thisset of amino acid residues forms the nucleotide label interactionregion. The specific molecular model for the binding of fluorescentlylabeled nucleotide to a DNA polymerase proposed by the applicants isused to predict the amino acid residues that form the nucleotide labelinteraction region of a given DNA polymerase. Applicants model for aconformational shift in DNA polymerase during DNA synthesis is offeredas a explanation of how the nucleotide label interaction region wasdetermined. The model provides guidance in making mutations in DNApolymerase that reduce the ability of a DNA polymerase to discriminateagainst the incorporation of fluorescently labeled nucleotides intopolynucleotides. FIG. 1 is a computer model showing how DNA and Taq DNApolymerase interact in the model. Whether or not the true mechanism ofDNA polymerase-nucleotide interaction is the same or different as themodel used to determine the parameters of the nucleotide labelinteraction region is not determinative to the operability of theinvention described herein.

The mutant DNA polymerases of the invention exhibit reduceddiscrimination against nucleotides labeled with a fluorescein-type dye.In other words, the mutant DNA polymerases of the invention contain atleast one mutation that increases the ability of the polymerase toincorporate a fluorescein-type dye labeled nucleotide relative to thecorresponding unlabeled nucleotide. In addition to reduceddiscrimination against nucleotides labeled with fluorescein-type dyes,the mutant DNA polymerases of the invention may also exhibit reduceddiscrimination against nucleotides labeled with other fluorescent dyesthat are not fluorescein-type dyes, as well as reduced discriminationagainst other detectable moieties. The fluorescently labeled nucleotidesfor which a given embodiment of the mutant DNA polymerases of theinvention exhibit reduced discrimination may vary with respect to theparticular fluorescent label, the linker used to attach the fluorescentlabel to the nucleotide, the site of attachment for the linker on thefluorescent label, the specific nucleotide base that is selected, andthe site of attachment for the linker on the nucleotide. The precisedegree of reduction in discrimination against a fluorescently labelednucleotide will vary in accordance with the specific mutation ormutations introduced into the DNA polymerase. The precise degree ofreduction in discrimination will also vary in accordance with thespecific fluorescently labeled nucleotide assayed, e.g., variations inbase, dye, or linker. Mutant DNA polymerase of the invention may exhibitanywhere from a slight reduction in discrimination against fluorescentlylabeled nucleotides to a complete elimination in discrimination, i.e.,the mutant enzyme does not significantly differ with respect of rate ofincorporation of labeled or unlabeled nucleotides. It is preferable touse embodiments of the subject mutant DNA polymerases that have at leasta two-fold reduction in discrimination against one or more fluoresceintype dye labeled nucleotides.

It will be appreciated by persons skilled in the art of molecularbiology that the nucleotide label interaction region of a given DNApolymerase is defined with respect to a specific fluorescently labelednucleotide. Changes in one or more of the following parameters of thestructure of a fluorescently labeled nucleotide may alter the identityof the amino acid residues that form the nucleotide label interactionsite of a given DNA polymerase: (1) identity of the base, (2) the siteof attachment on the nucleotide base, (3) the identity of the linkerjoining the base to the florescent dye, and (4) the identity of thefluorescent dye. The nucleotide labeled interaction region of Taqdefined with respect to TET(II)•ddCTP comprises the amino acid residuesE520. A53 1, L522, R523, E524, A525, H526, P527, I 528, V529, E530,K531, I532, R536, E537, R573, Q582, N583, V586, R587, P589, Q592, R593,R595, D610, T612, Q613, E615, R636, D637, T640, F647, V654, D655, P656,L657, R659, R660, T664, E681, L682, A683, I684, P685, E688, F692, Q754,H784, L817, E820, L828, K831, and E832. The sites at R660, T664, andE681 are of prefered sites for introducing mutations. Given that the3-dimensional structure of Taq DNA polymerase (and other DNApolymerases) is well known and the three dimensional structure ofTET(II)•ddCTP is understood with a high degree of certainty, thelocation of the amino acid residues that constitute the labelednucleotide interaction region with respect to TET(II)•ddCTP may betranslated to a different set of amino acid residues to accommodatestructural differences between TET(II)•ddCTP and other fluorescentlylabeled nucleotides so as to define the labeled nucleotide interactionsite with respect to those other nucleotides. For example, increasingthe length of the linker between the base and the fluorescent label andthe base may predictably alter the identity of amino acid residues thatform the labeled nucleotide interaction site, even though the base, baseattachment site, and fluorescent dye are the same. In many embodimentsof the subject polymerases, the set of amino acid residues that form thelabeled nucleotide interaction site with respect to a givenfluorescently labeled nucleotide will overlap with the set of amino acidresidues that form the labeled nucleotide interaction site as definedwith respect to a second fluorescently labeled nucleotide.

Embodiments of the invention include mutant DNA polymerases that exhibitreduced discrimination against nucleotides labeled with fluorescein-typedyes, wherein the fluorescein type dye is joined to the nucleotide baseby an alkynylamino-type linker. The fluorescein-type dye may be afluorescent energy transfer dye, comprising a fluorescein-type dyemoiety as a component of the energy transfer dye. In addition to reduceddiscrimination against fluorescently labeled nucleotides comprising analkynylamino-type linker, the mutant DNA polymerases of the inventionmay also exhibit reduced discrimination against nucleotides comprisingother types of linker. In order to minimize stearic interference betweenthe polynucleotide and the fluorescent label, purines are usuallylabeled at position 7 and pyrimidines are usually labeled at position 5.

Mutant DNA polymerases of the invention have one or more discriminationreducing mutations at amino acid residue positions within the nucleotidelabel interaction region of a given DNA polymerase. Discriminationreducing mutations are usually, although not necessarily, substitutionmutations. Several different amino residues may be substituted at agiven position of a parent enzymes so as to give rise to adiscrimination reducing mutations. The amino acid residues at a givenresidue position within the nucleotide label interaction region may besystematically varied so as to determine which amino acid substitutionsresult in the reduction of discrimination against the fluorescein-typedye labeled nucleotide dye of interest and the degree of such areduction in discrimination. The extent to which a particular mutation(or set of mutations) reduces discrimination may be measured by aselectivity assay as described in example 2. The substitution mutationis preferably, although not necessarily, a mutation that reduces thesize of the amino acid residue side chain of the amino acid residuepresent in the parent DNA polymerase. Mutations are preferably, althoughnot necessarily, conservative so as to maintain the specific polar ornon-polar character of the amino acid residue at the analogous positionparent molecule. The mutations in the nucleotide label interactionregion of a DNA polymerase preferably result in the substitution of theamino acid residue of the parent enzyme with the amino acid residue atthe corresponding position of phage T7 DNA polymerase (provided that adifference exists between the amino acid residues at that position in T7polymerase and the parent enzyme).

Discrimination reducing mutations are in the nucleotide labelinteraction region of DNA polymerases. The nucleotide label interactionregion is formed by portions of three regions of the DNA polymerase.These three regions are located in (i)the O-helix, (ii) the K helix, and(iii) the inter O—P helical loop of Taq DNA polymerase or analogouspositions in other DNA polymerases. Positions in Taq DNA polymerase thatform the nucleotide label interaction region are positions E520. A531,L522, R523, E524, A525, H526, P527, I 528, V529, E530, K531, I532, R536,E537, R573, Q582, N583, V586, R587, P589, Q592, R593, R595, D610, T612,Q613, E615, R636, D367, T640, F647, V654, D655, P656, L657, R659, R660,T664, E681, L682, A683, I684, P685, E688, F692, Q754, H784, L817, E820,L828, K831, and E832. Analogous positions in DNA polymerases other thanTaq are also form a nucleotide label interaction region. Preferredpositions for substitution mutations are R595, D655, R660, and E681. Aparticularly preferred position for mutations is E681, with thepreferred substitution at position 681 being M. Other suitablesubstitution mutations at E681 are as follows (listed in order ofdecreasing preference, excpt where note by a equal sign to denoteapproximate equivalence”): M>I>W>L>V>P>H=K=G=T=S>D=A=N>Y=C. A preferredsubstitution mutation at position R660 is R660D.

The specific amino acid residues that form the nucleotide interactionregion will vary in accordance with the particular DNA polymeraseselected as a parent enzyme for the introduction of discriminationreducing mutations. The determination of analogous amino acid residuespositions between different DNA polymerases may easily be achieved bythe person skilled in the art because of the large number of DNApolymerase amino acid sequences that have been determined and the manyregions of homology have been found between these different DNApolymerases. For example, a large compilation of the amino acidsequences of DNA polymerases from a wide range of organism and homologyalignments between the sequences can be found in Braithwaite and Ito,Nucl. Acids Res. 21(4):787-802 (1993). Examples of amino acid residueswithin the nucleotide label interaction regions of phage T7 polymeraseand E. coli DNA polymerase are provided in Table 1. In addition toproviding mutant DNA polymerases having reduced discrimination forfluorescein type dyes in Taq, T7 and E. coli DNA polymerase I, theinvention provides mutant DNA polymerases from many other organisms. Ingeneral, the teachings of the invention may used to produce mutant DNApolymerases having reduced discrimination for fluorescein type dyes fromany DNA polymerase that shares sufficient amino acid sequence homologyto Taq DNA polymerase to permit a person of ordinary skill in the art toidentify one or more amino acid residue positions in the DNA polymerasethat are analogous to positions E520. A531, L522, R523, E524, A525,H526, P527, I528, V529, E530, K531, I532, E537, R573, V586, R587, P589,Q592, R593, R595, D610, T612, Q613, E615, R636, T640, F647, V654, D655,P656, L657, R659, R660, T664, E681, L682, A683, I684, P685, E688, F692,Q754, L817, E820, L828, K831, and E832 in Taq DNA polymerase. Parent DNApolymerases that may be modified to contain discrimination reducingmutations in the nucleotide label interaction region include, but arenot limited to, DNA polymerases from organisms such as Thermus flavus,Pyrococcus furiosus, Thermotoga neapolitana, Thermococcus litoralis,Sulfolobus solfataricus, Thermatoga maritima, E. coli phage T5, and E.coli phage T. The DNA polymerases may be thermostable or notthermostable. It will be appreciated that the present invention enablespersons skilled in the art to introduce fluorescein-type dyediscrimination reducing mutations in to DNA polymerases from a widevariety of organisms, including DNA polymerases that have not beenisolated at the time of the filing of this application provided.Additionally, embodiments of the invention includes some purifiednaturally-occurring DNA polymerases that have the desired low degree ofdiscrimination against fluorescently labeled nucleotides. Suchnaturally-occurring DNA polymerases are structurally and functionallyanalogous to the mutant DNA polymerases explicitly described herein.

The amino acid residues that constitute the nucleotide label interactionregion of a given DNA polymerase vary in accordance with the specificfluorescently labeled nucleotide that is used to define the nucleotidelabel interaction region. Similarly, the mutations that arediscrimination reducing mutations may vary in accordance with thespecific fluorescently labeled nucleotide that is used to define thelabeled nucleotide interaction region. Additionally, the degree ofdiscrimination reduction achieved by the mutation (or mutations) in thelabeled nucleotide interaction site may vary with the specific labelednucleotide of interest. For example, E681M is the preferreddiscrimination reducing mutation in Taq with respect to TET(II)•ddCTPresulting in a 47× reduction in discrimination and a significantly lowerreduction in discrimination against a second fluorescently labelednucleotide. Conversely, an E681T mutation may result in a high levelreduction in discrimination against the second fluorescently labelednucleotide and only a low level of reduction in discrimination againstTET(II)-•dCTP.

Given that a mutant DNA polymerase of the invention may havediscrimination reducing mutation in the nucleotide label interactionregion resulting in a significant degree of reduction in discriminationfor a specific fluorescently labeled nucleotide and little or noreduction in the degree of reduction of discrimination against anotherfluorescently labeled nucleotide (assuming there is significantdiscrimination against that fluorescently labeled nucleotide by theparent DNA polymerase), a given mutant DNA polymerase may be said to be“receptive” with respect to one or more given fluorescently labelednucleotide. A specific mutant DNA polymerase is referred to as“receptive” with respect to a specific fluorescently labeled nucleotideif a discrimination reducing mutation in the nucleotide labelinteraction site in the specific enzyme of interest results in at leasta five fold reduction in discrimination against that given fluorescentlylabeled nucleotide. A mutant DNA polymerase of the invention may bereceptive with respect to more than one fluorescently labelednucleotide. Conversely, a specific fluorescently labeled nucleotide maybe “receptive” with respect to a given mutant DNA polymerase of theinvention.

In embodiments of the subject mutant DNA polymerases comprising morethan, one discrimination reducing mutation in the nucleotide labelinteraction region, the mutation site may be in the same or differentregion of the three regions of a polymerase that form the nucleotidelabel interaction region. In general, mutant DNA polymerases of theinvention will have 1, 2, or 3 discrimination reducing mutations.However, the invention also provides mutant DNA polymerases having morethan 3 discrimination reducing mutations. By combining multiplediscrimination reducing mutations, greater levels of reduction inlabeled nucleotide discrimination may be achieved. However, in manyembodiments of the invention, mutant DNA polymerases have levels ofreduced labeled nucleotide discrimination that are the same or less thanthe levels of DNA polymerase with single discrimination reductionmutations in the nucleotide label interaction region. Preferredcombinations of mutations in a Taq DNA polymerase background are R660D,E681G, and F667Y, i.e., Taq DNA polymerase mutant (R660D, E681G, andF667Y).

Different embodiments of DNA polymerase having mutations in thenucleotide label interaction region differ with respect to the degree ofreduction in discrimination against specific fluorescently labelednucleotides. These differences may be measured by an assay in order todetermine which specific embodiments have the greatest degree ofreduction in discrimination against the particular fluorescently labelednucleotides of interest. Generally, such assays measure competitionbetween a fluorescently labeled nucleotide and an unlabeled nucleotidefor incorporation into the same site on a primed template. One exampleof such an assay (referred to herein as a “selectivity assay”) isdescribed in detail below in Example 2.

The mutant DNA polymerases of the invention may comprise numerousmutations in addition to discrimination reduction mutations in thenucleotide label interaction region. These secondary mutations may beeither inside or outside the nucleotide label interaction region.Secondary mutations may be selected so as to have as to confer someuseful property on the mutant DNA polymerase. For example, additionalmutations may be introduced to increase thermostability;

decrease thermostability, increase processivity, decrease processivity,decrease 3′-5′ exonuclease activity, increase 3′-5′ exonucleaseactivity, decrease 5′-3′ exonuclease activity, increase 5′-3′exonuclease activity, and increase incorporation of dideoxynucleotides.Alternatively, the secondary mutations may be essentially neutral inknown effect.

Of particular interest are embodiments of the subject mutant DNApolymerase that comprise one or more secondary mutation that reduce3′-5′ exonuclease activity. DNA polymerases that are deficient in 3′-5′exonuclease activity have superior properties for PCR and for chaintermination 10 polynucleotide sequencing. Mutations that reduce 3′-5′exonuclease activity in DNA polymerase are well known to person ofordinary skill in the art. Detailed guidance on how to introducemutations that reduce 3′-5′ exonuclease activity can be found, amongother places in U.S. Pat. No. 4,795,699 (Tabor); U.S. Pat. No.5,541,099; U.S. Pat. No. 5,489,523; and Bernad et al., Cell 59:219-288(1989). Examples of such mutations in Taq DNA polymerase include G46D.For embodiments of the mutant DNA polymerases that are used forsequencing, it is preferable to include a G46D (or analogous mutationsin DNA polymerases other than Taq) in addition to mutations in thenucleotide label interaction region.

Also of interest among secondary mutations in the subject DNA polymerasemutants are mutations that increase incorporation of dideoxynucleotides,i.e., reduce the ability of a DNA polymerase to discriminate againstdideoxynucleotide as opposed to deoxynucleotides. Guidance on makingsuch mutations can be found, among other places in published PCTapplication WO96/12042 (application number PCT/US95/12928). Ofparticular interest is the mutation F667Y in Taq and analogous mutationsin other DNA polymerase. While F667Y is not part of the nucleotide labelinteraction region in Taq DNA polymerase with respect to Tet(II)•ddLTP,F667Y mutations may reduce discrimination against fluorescein-type dyelabeled nucleotides (see Table 1). Accordingly, for use in certainprocedures, e.g., DNA sequencing, be desirable to combine an F667Ymutations with one or more discrimination reducing mutations in thenucleotide label interaction region so as to reduce discrimination ofthe polymerase between deoxynucleotides and 2′3′ dideoxynucleotides.Mutant DNA polymerase of the invention having the F667Y mutation (orequivalent thereof) are particularly useful in Sanger type DNAsequencing with fluorescently labeled 2′3′ dideoxynucleotide chainterminators.

Numerous genes encoding DNA polymerases have been isolated andsequenced. This sequence information is available on publicly accessibleDNA sequence databases such as GENBANK. A large compilation of the aminoacid sequences of DNA polymerases from a wide range of organism can befound in Braithwaite and Ito, Nucl. Acids Res. 21(4):787-802 (1993).This information may be used in designing various embodiments of DNApolymerases of the invention and polynucleotide encoding these enzymes.The publicly available sequence information may also be used to clonegenes encoding DNA polymerases through techniques such as geneticlibrary screening with hybridization probes.

Other embodiments of the invention are polynucleotide sequences encodingthe mutant DNA polymerases provided herein. Polynucleotide sequencesencoding the mutant DNA polymerase of the invention may be used for therecombinant production of the mutant DNA polymerases. Polynucleotidesequences encoding mutant DNA polymerases having reduced discriminationagainst fluorescently labeled nucleotide may be produced by a variety ofmethods. A preferred method of producing polynucleotide sequencesencoding mutant DNA polymerases having reduced discrimination againstfluorescently labeled nucleotides is by using site-directed mutagenesisto introduce desired discrimination reducing mutations intopolynucleotides encoding the parent DNA polymerase molecules.Site-directed mutagenesis techniques are well known in the art asexemplified by U.S. Pat. No. 4,711,848; U.S. Pat. No. 4,873,192; U.S.Pat. No. 5,071,743; U.S. Pat. No. 5,284,760; U.S. Pat. No. 5,354,670;U.S. Pat. No. 5,556,747; Zoller and Smith, Nucleic Acids Res.10:6487-6500 (1982), and Edelman et al DNA 2:183 (1983). Detailedprotocols for site-directed mutagenesis are also given many generalmolecular biology textbooks such as Sambrook et al Molecular Cloning aLaboratory Manual 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor(1989), Ausubel et al. Current Protocols in Molecular Biology, (currentedition). Additionally, many text books on PCR (the polymerase chainreaction), such as Diefenbach and Dveksler, PCR Primer: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1995),describe methods of using PCR to introduce directed mutations. Genesencoding parent DNA polymerase may be isolated using conventionalcloning techniques in conjunction with publicly-available sequenceinformation. Alternatively, many cloned polynucleotide sequencesencoding DNA polymerases have been deposited with publicly-accessiblecollection sites, e.g., the American type culture collection depositaccession number ATCC 40336 is a phage clone of Taq DNA polymerase.

In addition to producing the mutant DNA polymerase encodingpolynucleotides of the invention by introducing directed mutations intopolynucleotides encoding parent DNA polymerases, it is possible(although difficult) to produce the polynucleotides of the inventionprimarily by in vitro DNA synthesis techniques. In vitro DNA synthesistechniques are well known to those skilled in the art and examples of invitro DNA synthesis can be found in U.S. Pat. No. 5,252,530; U.S. Pat.No. 4,973,679; U.S. Pat. No. 5,153,319; U.S. Pat. No. 4,668,777; U.S.Pat. No. 4,500,707; U.S. Pat. No. 5,132,418; U.S. Pat. No. 4,415,732;U.S. Pat. No. 4,458,066; and U.S. Pat. No. 4,811,218. When producingrelative polynucleotide molecules by in vitro DNA synthesis, smallermolecules are usually produced first and subsequently joined together byhybridization and ligation. Mutant DNA polymerase encodingpolynucleotides may also be produced by a combination of in vitrosynthesis and site-directed mutagenesis of cloned genes.

Polynucleotide encoding the mutant DNA polymerase of the invention maybe used for the recombinant expression of the mutant DNA polymerases.Generally, the recombinant expression of the mutant DNA polymerase iseffected by introducing a mutant DNA polymerase into an expressionvector adapted for use in particular type of host cell. Thus, anotheraspect of the invention is to provide expression vectors comprising apolynucleotide encoding a mutant DNA polymerase of the invention, suchthat the polymerase encoding polynucleotide is functionally inserted intthe expression vector. The invention also provide host cells comprisingthe expression vectors of the invention. Host cells for recombinantexpression may be prokaryotic or eukaryotic. Example of host cellsinclude bacterial cells, yeast cells, cultured insect cell lines, andcultured mammalian cells lines. Preferably, the recombinant host cellsystem is selected so as to closely match the organism from which themutant DNA polymerase was derived. For example, prokaryotic DNApolymerases are preferably expressed in a prokaryotic expression system.A wide range of expression vectors are well known in the art.Description of various expression vectors and how to use them can befound among other places in U.S. Pat. No. 5,604,118; U.S. Pat. No.5,583,023; U.S. Pat. No. 5,432,082; U.S. Pat. No. 5,266,490; U.S. Pat.No. 5,063,158; U.S. Pat. No. 4,966,841; U.S. Pat. No. 4,806,472; U.S.Pat. No. 4,801,537; and Goedel et al., Gene Expression Technology,Methods of Enzymology Vol. 185, Academic Press, San Diego (1989). Theexpression of DNA polymerases in recombinant cell systems is awell-established technique. Examples of the recombinant expression ofDNA polymerase can be found in U.S. Pat. No. 5,602,756; U.S. Pat. No.5,545,552; U.S. Pat. No. 5,541,311; U.S. Statutory Inventor RegistrationH1,531; U.S. Pat. No. 5,500,363; U.S. Pat. No. 5,489,523; U.S. Pat. No.5,455,170; U.S. Pat. No. 5,352,778; U.S. Pat. No. 5,322,785; and U.S.Pat. No. 4,935,361.

Other embodiments of the invention include multiple DNA polymerasecompositions particularly useful for polynucleotide sequencing, suchcompositions comprise at least two different mutant DNA polymerases ofthe invention, wherein (1) the first mutant DNA polymerase is receptivewith respect to a first fluorescently labeled nucleotide; (2) the secondmutant DNA polymerase is receptive with respect to a secondfluorescently labeled nucleotide; and (3) the first and secondfluorescently labeled nucleotides differ from one another with respectto their nucleotide bases and fluorescent labels. The first and secondfluorescently labeled bases may also differ with respect to one anotherby way of the linker, the base attachment position, or the fluorescentdye attachment site. The subject compositions are useful for catalyzingthe sequencing reactions in Sanger type DNA sequencing with fluorescentdye labeled 2′3′ dideoxy chain terminating nucleotides. Chaintermination sequencing with fluorescently labeled terminators preferablyemploys at least two, and more preferably 4 different fluorescentlylabeled chain terminators, wherein each different base is labeled with adistinctive fluorescent label. Because of the necessary structuraldifferences between the different fluorescently labeled chainterminators required for a sequencing reactions, i.e., nucleotide basesand fluorescent labels, there are many mutant DNA polymerases of theinvention that are not receptive to all of the fluorescently labeledterminators necessary for a given sequencing reaction. Thus, there areembodiments of the subject DNA polymerases that may have undesirablyhigh levels of discrimination against one or more of the labeledterminators used in a sequencing reaction set. The subject compositionsof two or more mutant polymerases ameliorates this problem bysimultaneously employing multiple mutant DNA polymerases that arereceptive to different chain labeled terminators, thereby having atleast one of the mutant polymerases “compensate” for the discriminationagainst a particular fluorescently labeled terminator by the otherpolymerases catalyzing the sequencing reactions. The ratio of thedifferent DNA polymerases in the composition preferably are selected soas to result in approximately equal levels of total activity for each ofthe different mutant DNA polymerases. Differences in specific activitybetween the different mutant polymerases may be taken into account whenequalizing total activity ratios between the polymerases. Differences inactivity levels between the various mutant DNA polymerases in thesubject compositions may also be compensated for by adjusting the levelsof the different fluorescently labeled terminators in the subjectcompositions. The subject multiple polymerase compositions may comprisetwo, three, four, or more different mutant DNA polymerases. The mutantpolymerase may or may not be derived from the same species or strain.The different mutation DNA polymerases in the subject mutant polymerasecompositions may or may not be receptive for one or more of thefluorescently labeled nucleotides in a given set fluorescently labeleddideoxynucleotides for sequencing.

The invention also includes various methods of using the mutant DNApolymerases (or subject multiple mutant DNA polymerase compositions) ofthe invention. The mutant DNA polymerases of the invention may besubstituted for the corresponding parent DNA polymerases in mostprocedures that employ DNA polymerases. In order to more fully takeadvantage of the properties of the subject mutant DNA polymerases, theamount (or concentration) of labeled and unlabeled nucleotides used inthe methods of the invention may be changed with respect to the amounts(or concentrations) used in the corresponding methods employingconvention DNA polymerases. These changes in the amount of nucleotidemay be optimized by routine experimentation. Methods of the inventioncomprise the step of extending a primed polynucleotide template with atleast one fluorescently labeled nucleotide, wherein the extension iscatalyzed by a mutant DNA polymerase of the invention. Thus, the subjectmethods result in the formation of one or more different fluorescentlylabeled polynucleotides produced by primer extension. The subjectmethods of synthesizing a fluorescently labeled polynucleotide may beused in a variety of procedures including, but not limited to, Sangersequencing (e.g., dideoxy nucleotide chain termination), the polymerasechain reaction (PCR), polynucleotide labeling, minisequencing. Thereduced discrimination against fluorescently labeled nucleotideproperties of the subject mutant DNA polymerase is particularly usefulfor Sanger DNA sequencing reactions, including cycle sequencing. The useof the subject mutant DNA polymerases for Sanger sequencing reduces theamount of fluorescently labeled chain terminating nucleotides requiredfor a sequencing reaction an may in many case be used to increase thenumber of bases that may be identified in single sequencing reactionthat is analyzed on an automated fluorescence-based sequencing apparatussuch as an Applied Biosystems 310 or 377(Applied Biosystems Division ofPerkin-Elmer, Foster City, Calif.). Detailed protocols for Sangersequencing are known to those skilled in the art and may be found, forexample in Sambrook et al, Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989).

The invention also provides kits for synthesizing fluorescently labeledpolynucleotides. The kits may be adapted for performing specificpolynucleotide synthesis procedures such as DNA sequencing or PCR. Kitsof the invention comprise a mutant DNA polymerase of the invention and afluorescently labeled nucleotide that exhibits reduced discriminationwith respect to the mutant DNA polymerase in the kit. Kits preferablycontain detailed instructions on how to perform the procedures for whichthe kits are adapted. Optionally, the subject kit may further compriseat least one other reagent required for performing the method the kit isadapted to perform. Examples of such additional reagents includeunlabeled nucleotides, buffers, cloning vectors, restrictionendonucleases, sequencing primers, and amplification primers. Thereagents include in the kits of the invention may be supplied inpremeasured units so as to provide for greater precision and accuracy.

Other embodiments of the invention include kits comprising (1) thesubject compositions of multiple mutant DNA polymerases, and (2)fluorescently labeled chain terminating nucleotides suitable for usewith the subject compositions, i.e., each labeled chain terminator isreceptive with respect to at least one of the mutant DNA polymerases inthe composition. Additional embodiments of the invention include kitsfor sequencing DNA that comprise a multiple mutant polymerasecomposition of the invention and at least two different fluorescentlylabeled chain terminating nucleotides are labeled at different bases,wherein each of the fluorescently labeled chain terminating nucleotidesis receptive with respect to at least one mutant DNA polymerase in thecomposition.

The invention, having been described above, may be better understood byreference to the following examples. The examples are offered, for amongother reasons, to illustrate specific embodiment of the invention andshould not be construed as a limitation on the invention.

EXAMPLES Example 1

Purification of Mutant Forms of Tag DNA Polymerase

Lysates of E. coli containing recombinant constructs designed for theproductiion of recombinant mutant Taq DNA polymerases were madeessentially as described in tDesai, U. J. and Pfaffle, P. K.,Biotechniques, 19:780-784 (1995). In order to prevent the polymerasefrom binding to chromosomal and plasmid DNAs contaminating the lysate, 5M NaCl was added dropwise to the heat treated, clarified lysates tobring the final NaCl concentration to 0.25 M. DNA was then precipitatedfrom this mixture by dropwise addition of 5% polyethylimine (in 20 mMTRIS.CL, pH 8.5) to make the final concentration of PEI 0.3%.Precipitation was allowed to continue for 5 minutes on ice. A white,cloudy precipitate was removed by centrifugation at 15,000×g for 15minutes at 4° C. The supernatant fluid was decanted and saved. Followingcentrifugation, the NaCl concentration was reduced to 0.13 M bymonitoring conductivity of the solution during the addition of TETTminus NaCl (20 rim TRIS.Cl, 0.1 mM EDTA, 0.05% Tween-20, 0.05%Triton-X100, 1% glycerol, pH 8.5).

Excess PEI was removed using a Bio-Rex 70 (BIO-RAD, Richmond, Calif.)column (2.5×30 cm). The column was poured and equilibrated with TETTBuffer+0.1 M NaCl. The polymerase does not bind to the Bio-Rex 70 underthese conditions.

To remove contaminating E. coli proteins, the Bio-Rex 70 column eluatewas loaded directly onto a Heparin-Agarose (Sigma Chemical Company, St.Louis, Mo.) column (1.5×30 cm) which was also poured and equilibrated inTETT Buffer+0.1 M NaCl. The heparin-agarose column was washed with 2column volumes of TETT+0.1 M NaCl and Taq DNA polymerase was eluted as asharp peak using TETT+1 M NaCl. Elution was monitored at 280 nm.

The heparin-agarose column fractions corresponding to the peakabsorbance were pooled and concentrated to 0.15 ml using Ultrafree-15Centrifugal Filter Devices (Millipore Corporation, MA) according to themanufacture's recommendations for centrifugation speeds and times. Theconcentrate was diluted to 15 ml with TETT Buffer+5% glycerol and thesample was re-concentrated to 0.15 ml. This was repeated one more timeto reduce the final NaCl concentration below 1 mM in the proteinsamples.

The concentrated polymerase samples were diluted two-fold using TETT+5%glycerol and an equal volume of TETT+95% glycerol was added to bring thefinal glycerol concentration to about 50%. Samples were stored at −20 °C. Protein concentrations were determined using the “Bradford ProteinAssay” (BIO-RAD, Richmond, Calif.). Activity was measured using aradiometric assay (described elsewhere).

Typical yields of polymerase from 2-liters of induced E. coli culture(corresponding to 30-50 ml of heat treated, clarified lysate) rangedfrom 4 to 24 mg. SDS-PAGE analysis of the purified samples showed onedark band of about 94,000 molecular weight) and several minor ones afterCoomassie Blue staining. The gels indicated a typical purification levelof >90%.

Example 2

Selectivity Assay

An unlabeled versus dye-labeled terminator assay (“terminator” isdefined as a non-extendible base such as 2′,3′-ddNTPs) was used toscreen mutant Taq DNA polymerase samples for better Tet(II)•ddCTPincorporating mutant forms of this polymerase. This assay is based upontwo substrates competing for the same active site at the same timeduring a steady state reaction in which only the polymeraseconcentration is limiting. Therefore, the assay measure the polymerase's“selectivity” for the unlabeled versus the fluorescein-labeledterminator. The DNA Primer/template used in this assay format is givenbelow: 5′->(FAM)-CCC TCG CAG CCG TCC AAC CAA CTC A           GGG AGC GTCGGC AGG TTG GTT GAG T G C CTC TTG TTT<-5′

The next template position following the 3′-end of the primer isindicated above by the bold and underlined G.

The reaction consisted of:

-   -   80 mM TRIS.Cl (pH 9.0 at 20° C.)    -   1000 nM DNA primer/template [5′-(FAM)25mer/36 G₁ template]    -   2 mM MgCl₂    -   50 μM TET(II)•ddCTP    -   1 μM ddCTP    -   0.25 Units of enzyme    -   40 μL reaction volume    -   60° C. reaction temperature

Samples (2 μL) were removed from the reaction mixture at pre-determinedtimes (typically, 20 second intervals for 0.25 Units of polymeraseactivity per μL) and added to ice cold 50 μL 0.5 M EDTA (pH 8.0). Timedaliquots were mixed and held on ice for further processing.

Samples of each time point were processed to remove excess,unincorporated TET(II)•ddCTP. Typically, 1.6 μL of each quenched samplewere added to 250 μL of 0.8 M LiCl plus 0.2 μg/ml E. coli tRNA. followedby 750 μL of 95% ethanol. After mixing, the nucleic acids were allowedto precipitate for 20 minutes at −20° C. The precipitates were recoveredby centrifugation using standard procedures. The supernatant fluid wasdiscarded and pellets were dissolved in 50 μL of 50% formamide. Gelsamples were heat treated (95° C. for 2 minutes) and 2 μL were loadedper sample lane on a 16% denaturing DNA sequencing gel. Gels were run onan Applied Biosystems Model 373 Sequencer using GeneScan FragmentAnalysis software to measure the amount of FAM fluorescence in the bandscorresponding to the 25-mer primer, the 26-mer product (indicating a ddCincorporation event) and the apparent “27-mer” product band (indicatinga TET(H)•ddC incorporation event).

The fluorescence signal in each of the bands was summed and the percentof signal in each band was used for further calculations as anormalization to avoid lane to lane loading differences. Energy transferfrom the %-FAM moiety present on the apparent “27-mer” product moleculesto the Tet(II) moiety on the newly incorporated 3′-base was notcorrected since all ratios were compared to “wild type” or Taq G46D.)The normalized fluorescent signals in the 26-mer and “apparent” 27-merproduct bands were corrected for the different concentrations of the twomolecules used in the reaction and the corrected values were plottedversus time. The velocity of incorporation for each substrate wasdetermined using least square fits to the data. The ratio ofddC/TET(II)•ddC incorporation rates is equal to the selectivity biasthat the sample polymerase shows for the unlabeled versus theTET(II)-labeled nucleotides and reflects the following relationship:v _(ddC) v _(Tet•ddC)=(k _(cat) /K _(M))_(ddC) [ddC]÷(k _(cat) /K_(M))_(Tet•ddC) [Tet(II)•ddC]

where:

-   -   v_(ddC)=velocity of ddC incorporation    -   V_(Tet(II)•ddC)=velocity of Tet(II)•ddC incorporation    -   k_(cat)=catalytic rate constant    -   K_(M)=nucleotide equilibrium binding constant    -   [ddC]=concentration of ddCTP in the reaction    -   [Tet(II)•ddC]=concentration of Tet(II)¥ddCTP in the reaction

In this assay format, “wild-type” Taq or (Taq G46D) showed a selectivitybias or ddC/Tet(II)•ddC number of about 85 to 1. Mutants showing lowerselectivity bias ratios were submitted to further testing. The Table 2below shows the results for a few of the mutants tested by way of a fewexamples: TABLE 2 Taq Selectivity Number WT/Mutant G46D 85 85/85 or 1G46D; R660D 8 85/8 or ≈ 10 G46D; R595E 28 85/28 or ≈ 3 G46D; F667Y 2885/28 or ≈ 3 G46D; E681G 40 85/40 or ≈ 2 G46D; D655L 40 85/40 or ≈ 2

Example 3

Next Nucleotide Rate Effect Assay

An additional kinetic step between “ground state” nucleotide binding orinitial collision and correct base pair formation and the group transferreaction would be expected to slow the polymerase dissociation rate froman Enz•DNA complex having a 3′-dideoxynucleotide in an assay termed the“Next Nucleotide Rate Effect” (Patel et al., 1991). This assay measuresthe steady state rate of incorporation of ddTTP (i.e., the enzyme islimiting) in the absence or presence of the next correct nucleotide. Theprimer template pair is shown below: 5′->(FAM)-CCC TCG CAG CCG TCC AACCAA CTC A           GGG AGC GTC GGC AGG TTG GTT GAG T A G GTC TTGTTT<-5′

The next template position is indicated by the bold, underlined A. Thenext template position beyond A is G. Under steady state reactionconditions, essentially all of the available polymerase is bound to theprimer/template. When ddTTP is present alone in solution, it isincorporated following binding to its template position, A. Additionalincorporation events require the polymerase to dissociate from theEnz•DNA complex and find another available primer/template that has notalready undergone and incorporation event. Hence the rate ofincorporation under these conditions is the dissociation rate of thepolymerase from the Enz•DNA complex. If the next correct nucleotide,dGTP or ddCTP, is also present in the reaction mixture, the dissociationrate of the polymerase from the Enz•DNA•ddCTP complex, for example, willbe slower if there is an additional kinetic step between the grouptransfer reaction that incorporated the ddTTP and an attempt by thepolymerase to incorporate ddCTP in a processive mode of synthesis. Thisslower rate of dissociation an be detected as a slower incorporationrate of ddTTP since no chemistry can occur once ddTTP and the polymerasecan no be processive despite the presence of another correct nucleotide.As shown in FIG. 2, the presence of the next correct nucleotide doesindeed slow the turnover or dissociation rate of the polymerase (TaqG46D; F667Y). FIG. 2 also shows that the presence of a fluorescein dyeon the next correct nucleotide (in this case, Tet(II)•ddCTP), appears toaccelerate the turnover rate. We interpret this to mean that thepolymerase is constantly undergoing a conformational change and that itcan attempt to undergo the change even in the absence of the nextcorrect nucleotide. However, the presence of a fluorescein dye on thenext correct nucleotide blocks the ability of the polymerase to undergosuch a change and thereby causes an immediate dissociation of the enzymefollowing the group transfer step for ddTTP incorporation. Hence, thefluorescein dye appears to accelerate the polymerase dissociation rateby eliminating a kinetic step (or steps) following the group transferreaction.

FIG. 3 shows the results for a Next Nucleotide Rate Effect assay for a“multiple” mutant form of Taq DNA polymerase, Taq G46D; R660D; F667Y;E681G. In this case, the presence of Tet(II) on the next correctnucleotide is “transparent” to the mutant polymerase. We interpret thisto mean that the mutant polymerase can indeed undergo the same kineticsteps following group transfer that “wild-type” versions of thispolymerase undergo. We also interpret these results to indicate that theF667Y mutation belongs in a different class than the R660D or E681Gmutations since Taq G46D; F667Y still shows a “fluorescein-effect” inthe “Next Nucleotide Rate Effect” assay, however, the multiple mutant,Taq G46D; R660D, F667Y; E681G, does not.

Typical assay conditions for the Next Nucleotide Effect assay were asfollows:

-   -   1000 nM primer/template DNA    -   80 mM TRIS.Cl (pH 9.0@20° C.)    -   2.4 mM MgCl₂    -   0.02 Units/μL polymerase activity    -   400 μM each nucleotide (when present)

Samples were taken and processed in the same manner as described under“Selectivity Assay.” In this case, it is possible to distinguish addC-incorporation event from a Tet(II)•ddC incorporation event by themigration rate of the resulting fragments in a 16% gel. Incorporation ofddC results in a “normal” 26-mer band that migrates as expected above orslower than the 25-mer primer. Incorporation of Tet(II)•ddC results inslower migration causing the band to migrate with an apparent sizeequivalent to a 27- or 28-mer.

Example 4

Analysis of Additional Mutants

Table 1, provided below, provides a summary of results obtained withselectivity assays performed with several different Taq mutants. Theanalogous site for the mutation in the enzymes E. coli DNA polymerase Iand phage T7 DNA polymerase are also noted. The term “FS” refers to aTaq DNA polymerase having a F667Y mutation.

REFERENCES

-   Barnes, W. M. (1992) The fidelity of Taq polymerase catalyzing PCR    is improved by an N-terminal deletion. Gene 112: 29-35.-   Brandis, J. W., Edwards, S. G. and Johnson, K. A. (1996) Slow rate    of phosphodiester bond formations accounts for the strong bias that    Taq DNA polymerase shows against 2′,3′-dideoxynucleotide    terminators. Biochemistry 35: 2189-2200.-   Desai, U. J. and Pfaffle, P. K. Single-step purification of a    thermostable DNA polymerase expressed in Escherichia coli.    Biotechniques 19: 780-784.-   Fersht, A. (1985) in “Enzyme Structure and Function,” W.H. Freeman    and Company, 2nd ed., pp. 111-112.-   Johnson, K. A. (1993) Conformational coupling in DNA polymerase    fidelity. Ann. Rev. Biochem. 62: 685-713.-   Patel, S. S., Wong, I., and Johnson, K. A. (1991) Pre-steady-state    kinetic analysis of processive DNA replication including complete    characterization of an exonuclease-deficient mutant. Biochemistry    30: 511-525.

INCORPORATION BY REFERENCE

This application incorporates all publications, patents, and patentapplication referenced herein in there entirety.

EQUIVALENTS

While the invention has been described and illustrated with reference tospecific embodiments, those skilled in the art will recognize thatmodifications and variations may be made without departing from theprinciples of the invention as described hereinabove and set forth inthe following claims. TABLE 1 TET(II)ddCTP/ ROX-ddCTP/ TAMRA-dTTP/ TaqPol I T7 [Final] *Units Spec. ddCTP ddCTP ddTTP No. Mutant Equiv. Equiv.μg/μl per μl Act. [Mut/Wt] [Mut/Wt] [Mut/Wt] 1 G46D 4.4 200 45.5 1* 1 1G46D (LS.1) 28.0 1950 69.6 G46D (LS.2) 78.3 6240 79.7 FS RMS 3 920 2G46D; F667W F762 Y526 3.6 5 1.4 1 1 (LS.1) F762 Y526 25.0 150 6.0 3G46D; R573E R668 R429 11.0 0 0.0

4 G46D; E615L E710 E480 4.8 0 0.0

5 G46D; E615D E710 E480 10.4 600 57.7 no activity

6 G46D; E615I E710 E480 7.0 140 20.0 no activity

7 G46D; R587K R682 “V443” 8.9 420 47.2 1 1 8 G46D; R573K R668 R429 9.3 0

9 G46D; L657T E752 T517 9.5 450 47.4 1 1 0.5 10 G46D; R587K R682 “V443”nd nd

11 G46D; Q754S Q849 Q615 13.0 0 0.0

12 G46D; E615K E710 E480 1.8 0 0.0

13 G46D; R573Q R668 R429 14.0 800 57.1 1 1 14 G46D; D655L T750 L515 11.3400 35.4 2 0.7 0.5 15 G46D; Q754K Q849 Q615 nd nd nd

16 G46D; R595K R690 H460 nd nd nd

17 G46D; K831M H928 H704 8.3 300 36.1 1 1 18 G46D; L682G L777 “I540” 5.5200 36.4 1 1 6 19 G46D; R659K R754 D519 22.9 150 6.6 no activity 20G46D; A683E N778 V541 12.8 900 70.3 1 21 G46D; Q754K Q849 Q615 5.0 0 0.0

22 G46D; R593H R688 E458 16.7 700 41.9 1 23 G46D; R595E R690 H460 23.550 2.1 3 24 G46D; A683V N778 V541 11.4 340 29.8 1 25 G46D; Q592A R687A457 nd nd nd

26 G46D; R660D R755 D519 13.3 190 14.3 T0 27 G46D; T640G R735 T507 13.0225 17.3 1 28 G46D; E681G Q776 “I540” 7.5 170 22.7 2 29 G46D; V654E V749E514 9.2 210 22.8 30 G46D; Q613E Q708 G478 15.2 71 4.7 31 G46D; D610AQ705 D475 16.6 0 0.0 32 G46D; E820K E917 E693 11.6 475 40.9 33 G46D;L817A L914 L690 15.2 470 30.9 34 G46D; I684G I779 G542 35 G46D; R660D;F667Y 19.1 179 9.4 10 36 G46D; R595D; R660D; F667Y 10.5 0 0.0

37 G46D; D655L; R660D; F667Y 18.8 228 12.1 10 38 G46D; R660D; F667Y;E681G 13.1 404 30.8 12 39 G46D; R595E; F667Y 9.5 0 0.0

40 G46D; T7 Loop JK 12.7 0 0.0

41 G46D; A582Q583 13.2 0 0.0

42 G46D; P656S S751 16.2 560 34.6 K731 40 lysates 22 act. 17 testedmutants 4 > “wt”*WT = 85

TABLE 2 Tet Selectivity TET(II) · ddCTP/ddCTP* Lysate # GenotypeSpecific Activity (Mutant/WT) R660- Mutants Acidic- Aspartic acid 29 CS;R660D 14 10*  38 FS; R660D 9 10 39 R595E; FS; R660D 0 nd 40 D655L; FS;R660D 12 10 41 FS; R660D; E681G 31 12 49 CS; R660D 41 nd Glutamic Acid51 FS; R660E; E681G 11  7 72 FS; R660E 1  7 Basic- Lysine 50 FS; R660K28  1** Histidine 101 FS; R660H 13  1 Imino- Proline 66 FS; R660P 8  1Aliphatic- Alanine 68 FS; R660A 4  4 Isoleucine 73 FS; R660I 5  0.9***Valine 90 FS; R660V 10  1 55 FS; R660V; E681G 1  1 Leucine 91 FS; G660L8  0.6*** 52 FS; R660L; E681G 28  1 Glycine 47 FS; R660G; E681G 18  6 78FS; R660G 8  2 Polar Uncharged- Glutamine 53 CS; R660Q 47  1 69 FS;R660Q 5  3 Serine 98 FS; R660S 16  7 Cysteine 93 FS; R660C 14  4Asparagine 97 FS; R660N 13  3 Threonine 96 FS; R660T 26  3 MethionineAromatic- Phenyalanine 92 FS; R660F 9  0.1*** Tyrosine 95 FS; R660Y 17 1 E681- Mutants Acidic- Aspartic acid 71 FS; E681D 9  4** Basic- Lysine75 FS; E681K 52  6 Arginine Histidine 86 FS; E681H 37  7 Imino- Proline74 FS; E681P 19  9 Aliphatic- Alanine 63 FS; E681A 13  6 Isoleucine 99FS; E681I 37 27 Valine 76 FS; E681V 110 10 Leucine 87 FS; E681L 22 14Glycine 48 FS; E681G 37  6 Polar Uncharged- Glutamine Serine 61 FS;E681S 12  5 Cysteine 88 FS; E681C 20  2 Asparagine 89 FS; E681N 40  4Threonine 81 FS; E681T 35  6 Methionine 85 FS; E681M 32 47 Aromatic-Phenyalanine Tyrosine 80 FS; E681Y 42  3 Tryptophan 84 FS; E681W 37 1711-05-97 *Ratio > 1 means improved TET(II) · ddCTP incorporation.enzyme. **Ratio = 1 means wild-type activity. ***Ratio < 1 meansactivity worse than wild-type. M > I > W > L > V > P > H=K=G=T=S > D=A=N > Y=C47  27  17  14  10  9   7 6 6 6 5   4 4 4   3 2 Tryptophan 94 FS; R660W8  1  D > E=S > C=A=Q=T =N> G > K=P=V=Y=W=H > I  = L >> F10   7 7   4 4 3 3 3  2   1 1 1 1 1     0.9  0.6  0.1 *Ratio > 1 meansimproved TET(II) · ddCTP incorporation. Must be “85” to be “transparentto the enzyme. **Ratio = 1 means wild-type activity. ***Ratio < 1 meansactivity worse than wild-type.

1-15. (canceled)
 16. A method of performing a chain terminatingsequencing reaction, said method comprising, forming a mixturecomprising a first DNA polymerase, a second DNA polymerase, that isdifferent in structure from the first DNA polymerase, a firstfluorescent dye labeled terminator, and a second fluorescent dye labeledterminator, wherein the first DNA polymerase is receptive with respectto a first dye-labeled nucleotide and the second DNA polymerase isreceptive with respect to a different dye-labeled nucleotide, andperforming a chain terminating sequencing reaction.
 17. The method ofclaim 16, wherein the first and second DNA polymerase are receptive withrespect to nucleotides that differ with respect to one another by anucleotide base.
 18. The method of claim 17 wherein the first and secondDNA polymerase are receptive with respect to nucleotides that differwith respect to one another by a fluorescent dye.
 19. The method ofclaim 18 wherein the first and second DNA polymerase are receptive withrespect to nucleotides that differ with respect to one another by afluorescent dye and a nucleotide base.
 20. The method according to claim16, wherein the chain terminator sequencing reaction is a cyclesequencing reaction.
 21. The method according to claim 20, wherein thesequencing reaction does not take place concurrently with a PCRamplification reaction.
 22. The method of claim 16, wherein the firstDNA polymerase and the second DNA polymerase are derived from the samenaturally occurring DNA polymerase.
 23. A method of performing a chainterminating sequencing reaction, said method comprising, forming amixture comprising a first DNA polymerase, a second DNA polymerase,first fluorescent dye labeled terminator, and a second fluorescent dyelabeled terminator, wherein the first and second DNA polymerase arereceptive with respect to nucleotides, and performing a chainterminating sequencing reaction, wherein the first and second DNApolymerase are receptive with respect to nucleotides that differ withrespect to one another by a fluorescent dye.
 24. The method of claim 23wherein the first and second DNA polymerase are receptive with respectto nucleotides that differ with respect to one another by a fluorescentdye and a nucleotide base.